Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia.

BACKGROUND: Mutations in epigenetic modifiers were reported in patients with acute myeloid leukaemia (AML) including mutations in DNA methyltransferase 3A gene (DNMT3A) in 20%-30% patients and mutations in isocitrate dehydrogenase 1/2 gene (IDH1/2) in 5%-15% patients. Novel studies have shown that mutations in DNMT3A and IDH1/2 influence prognosis, indicating an increasing need to detect these mutations during routine laboratory analysis. DNA sequencing for the identification of these mutations is time-consuming and cost-intensive. This study aimed to establish rapid screening tests to identify mutations in DNMT3A and IDH1/2 that could be applied in routine laboratory procedures and that could influence initial patient management. METHODS: In this study we developed an endonuclease restriction method to identify the most common DNMT3A mutation (R882H) and an amplification-refractory mutation system (ARMS) to analyse IDH2 R140Q mutations. Furthermore, we compared these methods with HRM analysis and evaluated the latter for the detection of IDH1 mutations. RESULTS: Of 230 samples from patients with AML 30 (13%) samples had DNMT3A mutations, 16 (7%) samples had IDH2 R140Q mutations and 36 (16%) samples had IDH1 mutations. Sensitivity assays performed using serial dilutions of mutated DNA showed that ARMS analysis had a sensitivity of 4.5%, endonuclease restriction had a sensitivity of 0.05% and HRM analysis had a sensitivity of 5.9%-7.8% for detecting different mutations. HRM analysis was the best screening method to determine the heterogeneity of IDH1 mutations. Furthermore, for the identification of mutations in IDH2 and DNMT3A, endonuclease restriction and ARMS methods showed a perfect concordance (100%) with Sanger sequencing while HRM analysis showed a near-perfect concordance (approximately 98%). CONCLUSION: Our study suggested that all the developed methods were rapid, specific and easy to use and interpret. HRM analysis is the most timesaving and cost-efficient method to rapidly screen all the 3 genes at diagnosis in samples obtained from patients with AML. Endonuclease restriction and ARMS assays can be used separately or in combination with HRM analysis to obtain more reliable results. We propose that early screening of mutations in patients with AML having normal karyotype could facilitate risk stratification and improve treatment options.

Molecular analysis of different FLT3-ITD mutations in acute myeloid leukemia.

Mutation of the FMS-like tyrosine kinase-3 (FLT3) gene occurs with a frequency of 20-25% in acute myeloid leukemia (AML). Different studies have reported conflicting results, stating the importance of the length, position and number of internal tandem duplications (ITDs) for prognostic significance. In the present study, FLT3-ITD mutations were found in 51 (23%) of 218 patients with AML. Using sequence analysis we categorized ITD integration sites according to functional regions of the FLT3 receptor. Median ITD size was 61 bp. The insertion site was strongly correlated with ITD size: more C-terminal located inserted fragments were significantly bigger. Our data confirm that FLT3-ITD mutations identify a subset of young patients with AML with normal cytogenetics but with inferior outcome. Patients with AML with mutation localization outside the juxtamembrane domain showed no correlation with worse prognosis. A high mutant/wild-type ratio appears to have a major impact on the prognostic relevance.

Mesenchymal stromal cells of myelodysplastic syndrome and acute myeloid leukemia patients have distinct genetic abnormalities compared with leukemic blasts.

Mesenchymal stromal cells (MSCs) are an essential cell type of the hematopoietic microenvironment. Concerns have been raised about the possibility that MSCs undergo malignant transformation. Several studies, including one from our own group, have shown the presence of cytogenetic abnormalities in MSCs from leukemia patients. The aim of the present study was to compare genetic aberrations in hematopoietic cells (HCs) and MSCs of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Cytogenetic aberrations were detected in HCs from 25 of 51 AML patients (49%) and 16 of 43 MDS patients (37%). Mutations of the FLT3 and NPM1 genes were detected in leukemic blasts in 12 (23%) and 8 (16%) AML patients, respectively. Chromosomal aberrations in MSCs were detected in 15 of 94 MDS/AML patients (16%). No chromosomal abnormalities were identified in MSCs of 36 healthy subjects. We demonstrate herein that MSCs have distinct genetic abnormalities compared with leukemic blasts. We also analyzed the main characteristics of patients with MSCs carrying chromosomal aberrations. In view of these data, the genetic alterations in MSCs may constitute a particular mechanism of leukemogenesis.

Clinical features and prognostic implications of TCF3-PBX1 and ETV6-RUNX1 in adult acute lymphoblastic leukemia.

BACKGROUND: The t(9;22) and t(4;11) chromosomal translocations, which generate the BCR-ABL and MLL-AF4 fusion genes, define high-risk subtypes of acute lymphoblastic leukemia in adults. However, the prognostic impact of other rarer fusion genes is less well established in adult acute lymphoblastic leukemia than in the childhood form. DESIGN AND METHODS: In the context of the German Multicenter Therapy Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) we used reverse transcriptase polymerase chain reaction to investigate 441 cases of BCR-ABL- and MLL-AF4-negative B-precursor acute lymphoblastic leukemia for the TCF3-PBX1 (E2A-PBX1) and ETV6-RUNX1 (TEL-AML1) fusion transcripts generated by the t(1;19)(q23;p13.3) and t(12;21)(p13;q22) translocations. Both are well-known molecular alterations in pediatric acute lymphoblastic leukemia in which they have favorable prognostic implications. RESULTS: We identified 23 adult patients with TCF3-PBX1 and ten with ETV6-RUNX1. In contrast to previous reports we found no significant difference in overall survival between TCF3-PBX1-positive and -negative patients. At 2 years after diagnosis all the ETV6-RUNX1-positive patients were alive and in continuous complete remission, but their long-term outcome was negatively affected by late relapses. TCF3-PBX1-positive patients exhibited a characteristic CD34(-)/CD33(-) and mostly cyIg(+) immunophenotype. ETV6-RUNX1 only occurred in patients under 35 years old and was associated with a significantly lower white blood count. CONCLUSIONS: In contrast to previous suggestions, adult patients with TCF3-PBX1-positive acute lymphoblastic leukemia do not appear to have a worse outcome than their negative counterparts. ETV6-RUNX1-positive patients had a very favorable performance status during the first few years but their long-term survival was negatively affected by late relapses. Both groups of patients are characterized by distinct clinicobiological features which facilitate their diagnostic identification.